Tumor Necrosis Factor, or more specifically Tumor Necrosis Factor-.alpha. (for ease of reference, unless otherwise indicated, "Tumor Necrosis Factor" or "TNF" when used herein refers to TNF-.alpha.), is a cytokine, primarily produced by stimulated macrophages. It exhibits not only a striking cytotoxicity against various tumour cells [Carswell et al., Procd. Nat. Acad. Sci., USA 72, 3666-3670, (1975)] but also plays a multiple role as a mediator of inflammation and the immune response [for an overview see Beutler and Cerami, Ann. Rev. Immunol. 7, 625-655 (1989); Bonavista and Granger (eds.) "Tumor Necrosis Factor: Structure, Mechanism of Action, Role in Disease and Therapy, Karger, Basel (1990)". The primary structure of human Tumor Necrosis Factor-.alpha. (hTNF-.alpha.) has been deduced from the nucleotide sequence of a cDNA which has been cloned and expressed in E. coli [Pennica et al., Nature 312, 724-729 (1984); Marmenout et al., Europ. J. Biochem. 152, 515-522 (1985); Wang et al., Science 228, 149-154 (1985); Shirai et al., Nature 313, 803-806 (1985)]. A striking homology in amino acid sequence (30%) was found between hTNF-.alpha. and human Lymphotoxin, often referred to as human Tumor Necrosis Factor-beta (hTNF-.beta.), a cytokine mainly produced by activated lymphocytes [Gray et al., Nature 312, 721-724 (1984); Fiers et al., Cold Spring Harbour Symp. 51, 587-595 (1986)].
hTNF-.alpha. with modified amino acid sequences, so called TNF-.alpha.-muteins, have also been described in various publications--for example Yamagishi et al., Protein Engineering 3, 713-719, (1990); Fiers in "Tumor Necrosis Factors: Structure, Function and Mechanism of Action"; Fiers et al. in Bonavista and Granger, pp. 77-81 (see above); Goh et al., (1991), "Structural and functional domains in human tumor necrosis factors." Prot. Engineering 4: 385-389; Kircheis et al., (1992), "Biological activity of mutants of human tumor necrosis factor-.alpha., "Immunology 76: 433-438; Van Ostade et al., (1991), "Localization of the active site of human tumor necrosis factor (hTNF) by mutational analyses," EMBO J. 10: 827-836; Van Ostade et al., (1993), "Human TNF mutants with selective activity on the p55 receptor," Nature 361: 266-269; Zhang et al., (1992), "Site-directed mutational analysis of human tumor necrosis factor-.alpha. receptor binding site and structure-functional relationship," J. Biol. Chem. 267: 24069-24075; and in Ito et al., (1991), "Novel muteins of human tumor necrosis factor .alpha., "Biochim. Biophys. Acta 1096: 245-252. In addition TNF-.alpha.-muteins have been the object of several patent applications, e.g. International Patent Applications Publ. Nos. WO 86/02381, WO 86/04606, WO 88/06625 and European Patent Applications Publ. Nos. 155,549; 158,286; 168,214; 251,037 and 340,333, and Deutsche Offenlegungsschrift Nr. 3843534.
Muteins of Lymphotoxin have also been disclosed in the art, e.g. in European Patent Applications Publ. Nos. 250,000; 314,094 and 336,383, as well as in the following two publications: Goh et al., (1991), "Aspartic acid 50 and tyrosine 108 are essential for receptor binding and cytotoxic activity of tumor necrosis factor beta (lymphotoxin)," Prot. Engineering 4: 785-791 and Wakabayashi et al., (1990), "Deletion of lysine 89 enhances the cytotoxicity and the receptor binding affinity of human lymphotoxin," J. Biol. Chem. 265: 7604-7609.
The biological effects of TNF are mediated via specific receptors, namely a receptor with an apparent molecular weight of 55 kD on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) (p55-TNF-R) and a receptor with an apparent molecular weight of 75 kD on SDS-PAGE (p75-TNF-R).
Both forms of TNF-receptors have been cloned, namely p55-TNF-R by Loetscher et al. [Cell 61, 351-359, (1990)] and p75-TNF-R for example by Dembic et al. [Cytokine 2, 53-58, (1990)] (for both receptors see also European Patent Application No. 90116707.2) and it was found more recently that both receptors bind not only TNF-.alpha. but also TNF-.beta. with high affinity [Schonfeld et al., J. Biol. Chem. 266, 3863-3869 (1991)].
It is well known in the art that on the basis of its biological activities TNF-.alpha. can be a valuable compound for the treatment of various disorders. For example TNF-.alpha., alone or in combination with interferon, can be an effective antitumor agent [Brouckaert et al., Int. J. Cancer 38, 763-769 (1986)]. However, its systemic toxicity is a major limitation to its wider therapeutic use [Taguchi T. and Sohmura Y., Biotherapy 3, 177-186 (1991)].
Human TNF-.alpha. (hTNF-.alpha.) and murine TNF-.alpha. (mTNF-.alpha.) bind with almost equal affinity to human p55-TNF-R and to human p75-TNF-R. It has, however, been shown that in mice human TNF-.alpha. (hTNF-.alpha.), only binds to the smaller mouse TNF receptor (murine p55-TNF-R). In mice hTNF-.alpha. is far less toxic than murine TNF-.alpha. (mTNF-.alpha.), which binds to both mouse receptors, mp55-TNF-R and mp75-TNF-R. For example, in C57B16 mice, the LD50 is about 10 .mu.g/mouse and 500 .mu.g/mouse with mTNF-.alpha. and hTNF-.alpha., respectively [Brouckaert et al., Agents and Actions 26, 196-198 (1989); Everaerdt, B. et al., Biochem. Biophys. Res. Comm. 163, 378-385 (1989); Lewis, M. et al., Proc. Natl. Acad. Sci. USA 88, 2830 (1991); Brouckaert, P., Libert, C., Everaerdt, B. and Fiers, W. (1992). "Selective species specificity of tumor necrosis factor for toxicity in the mouse." Lymphokine Cytokine Res. 11, 193-196]. Hence it was proposed that the p75-TNF-R plays a special role in systemic toxicity.
It also has been reported that proliferative signals can be mediated by hp75-TNF-R in human T lymphocytes (Gehr et al., J. Immunol. 149, 911, 1992; Tartaglia et al., Proc. Natl. Acad. Sci. USA 88, 9292, 1991).
Human Tumor Necrosis Factor muteins, showing a significant difference between their binding affinity to the human p75-Tumor-Necrosis-Factor-Receptor (hp75-TNF-R) and to the human p55-Tumor-Necrosis-Factor-Receptor (hp55-TNF-R), have been described in European Patent Application Publication Nos. 486 908, and 563 714 where hTNF muteins are disclosed which have retained binding activity to hp55-TNF-R, but have lost nearly all binding to hp75-TNF-R.